The intestine is exposed to bacterial flora, dietary antigens and potential pathogens. To prevent chronic inflammation, various cell populations keep the mucosal immune response in check. Interleukin 10 (IL-10) is known as a cytokine with anti-inflammatory properties. IL-10 and IL-10 receptor (IL-10R) deficient mice develop spontaneous colitis in the presence of a normal intestinal microflora. Recent reports identified IL-10 as a susceptible locus for the development of inflammatory bowel diseases (IBD). Moreover IL-10R gene variants were found in some patients with early-onset colitis. Many studies identify CD4+ T cell-derived IL-10 as a key mediator of intestinal immune homeostasis, however, we have found IL-10 derived from intestinal F4/80+ CD11b+ CD11cint macrophages acts directly on regulatory T cells (Treg) and maintains their suppressive function in a mouse model of colitis. This suggests that IL-10 from macrophages rather than T lymphocytes also could be critical. Therefore the experiments in this application will focus on the cell type in the intestine that produces IL-10. We will investigate its phenotype, it's possible mode of regulation, and its broad effects on mucosal immunity including regulatory T cell function, using genetic technologies, cellular immunology methods, bioinformatics technologies and in vivo disease models. In Aim 1, we will examine how IL-10 producing macrophages modulate immune responses using mice with cell type-specific targeted deletion of the Il10 gene. We will fully characterize the phenotype and function of the large intestinal IL-10 producing macrophages compared with IL-10 non-producing cells in the regulation of mucosal homeostasis (Aim 2). We believe that our experimental system has relevance to the possible development of macrophage-based immune therapy in the future. In summary, the proposed experiments build on our novel preliminary findings in order to achieve a deep understanding of the pathway leading to IL-10 production by intestinal macrophages, and the means by which gut homeostasis is regulated.